The long-term goal of the proposed research is to identify the key regions within the primary sequence of theCa2+ release channel (ryanodine receptor; RyR1) of mammalian skeletal muscle sarcoplasmic reticulum (SR) which determine: 1. Sensitivity to physiologic activating and inhibiting cations, and 2. The ability of the channel to sense physiologic changes in local redox potential. Hypothesis I: Structural determinants of the cation activator and inhibitor sites are coordinated by spatially separated amino acids within the RyR1 linear sequence. Conserved negative charges within one or more of five cytoplasmic domains in the C-terminal -1000 amino acids of RyR1 coordinate cation binding and activation, whereas it appears that interactions between the C-terminal and central domains of RyR1, especially sequences encompassing difference regions D1 & D3, are needed to engage proper cation inhibition of RyRI. Specific Aim 1. Define the structural determinants in RyR1 that influence its binding and sensitivity to Ca2+ and Mg2+ using site-specific mutations of C-terminal truncated channels and full length RyR1 along with RyR1/RyR3 chimera. Specific Aim 2. Define the changes in cation binding constants and the activation energy (Ea) associated with channel activation for RyR1s with mutations within their cation binding motifs. Specific Aim 3. To define how altered cation regulation of RyR1 impacts excitation-contraction coupling and excitation-coupled Ca2+ entry. Hypothesis II: Hyper-reactive sulfhydryls in the primary structure of RyR1 constitute a trans- SR redox gradient sensor. Specific Aim 1. To mutate the six hyper-reactive thiols that we have discovered in RyR1 and define their contribution to trans SR redox sensing behavior. Specific Aim 2. To establish mechanistic links between redox state and the ability of cations to regulate RyR1 activity. The proposed studies will provide new information concerning the molecular mechanisms by which microsomal calcium channels residing within the SR/ER membrane are regulated by two important physiological parameters, cations (Ca2+and Mg2+) and cellular redox state. It is likely that these mechanisms are conserved among the multi-member family of Ca2+ release channels and may be significant in identifying new clinical targets for the prevention and treatment of injury resulting from oxidative stress.